ABSTRACT The long term goal of this proposal is to develop interventions for diabetic gastroparesis, a syndrome of delayed gastric emptying predominantly affecting women. Gastric motility dysfunction is a common complication of diabetes and can involve pathogenic oxidative stress. In rodent models, reduced nitric oxide (NO) availability as a result of inhibition of neuronal nitric oxide synthase (nNOS) and increased reactive oxygen species (ROS) has been shown to play a detrimental role in gastric motility. The transcription factor nuclear factor (erythroid-derived-2)-like 2 (NRF2) regulates the expression of Phase II antioxidant and detoxification genes. NRF2 protein stability is mainly regulated by two E3 ubiquitin ligase adaptors: CUL3/Keap1 and CUL1/SCF/TrCP. Independent of CUL3/Keap1, the GSK3/TrCP axis influences the nuclear exclusion and inactivation of NRF2. Preliminary studies indicate that expression of NRF2 and its target genes, Gclc and Gclm, are suppressed while oxidative stress is elevated in a rodent model of diabetes. We have demonstrated that loss of NRF2 (Nfe2/2-/-) resulted in decreased levels of tetrahydrobiopterin (BH4, a critical cofactor for nNOS dimerization and enzyme activity) and that this led to oxidative stress, inhibition of nNOS, reduced NO levels & gastric nitrergic neuron (neurons synthesizes nNOS)motility, and delayed gastric emptying compared to age-matched wild-type mice. These data support the notion that loss of NRF2 expression in diabetes impairs antioxidant gene expression, which deregulates NO synthesis, thereby contributing to the development of gastroparesis. We provide evidence that sex hormone estradiol-17? (E2) mediated NRF2/nNOS expression and gastric motility is impaired in the onset of diabetes. In addition, we have shown that glycogen synthase kinase 3beta (GSK-3?) or E2 regulate the synthesis of NO in female gastric tissue. This data suggest that loss of NRF2 expression during hyperglycemia is a consequence of activation of a GSK-3?/CUL1/SCF/TrCP/RBX1 axis . Our central hypothesis is that nitrergic mediated gastric motility is regulated by NRF2. Specific Aim 1 will test the hypothesis that loss of NRF2 expression during hyperglycemia is a consequence of activation of a GSK-3?/CUL1/SCF/TrCP/RBX1 axis. Specific Aim 2 will test the hypothesis that restoration of NRF2 and NRF2-regulated antioxidant enzyme expression will result in normal gastric motility and gastric emptying; and Specific Aim 3. will test the hypothesis that NRF2 and nNOS mediated gastric motility are regulated by female sex hormones; 17?-estradiol (E2), progesterone and/or their gastric receptors in diabetic animals. These studies will shed light on the mechanisms of NRF2-mediated gastric nNOS motility function and thereby enhance our understanding of the pathophysiology of gastroparesis. The research outlined will potentially identify novel treatment options for diabetes-induced gastric dysmotility, particularly in females.